METHODS IN MOLECULAR BIOLOGY™ Series Editor John M Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Liver Stem Cells Methods and Protocols Edited by Takahiro Ochiya National Cancer Center Research Institute, Tokyo, Japan Editor Takahiro Ochiya, Ph.D Chief, Division of Molecular and Cellular Medicine National Cancer Center Research Institute 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045 Japan tochiya@ncc.go.jp ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-467-4 e-ISBN 978-1-61779-468-1 DOI 10.1007 /978-1-61779-468-1 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011942927 © Springer Science+Business Media, LLC 2012 All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) Preface A Brief Outline of the Aims and Target Audience of Liver Stem Cells The role of a putative stem cells and liver-specific stem cell in regeneration and carcinogenesis is reviewed in this book There is increasing evidence that there is a liver stem cell that has the capacity to differentiate into parenchymal hepatocytes or into bile ductular cells These stem cells may be activated to proliferate after severe liver injury or exposure to hepatocarcinogens Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration In this book, we focus on recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries Several types of stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, haematopoietic stem cells, and mesenchymal stem cells, can be induced to differentiate into hepatocyte-like cells in vitro and in vivo Stem cell transplantation has been shown to significantly improve liver function and increase survival in experimentally induced liver-injury models in animals Furthermore, several pilot clinical studies have reported encouraging therapeutic potential of stem cell-based therapies This book consists of five main categories: (1) Several hepatic progenitor cells; (2) Hepatic differentiation from stem cells; (3) Bile ductal cell formation from stem cells; (4) Liver stem cells and hepatocarcinogenesis; and (5) Application of liver stem cells for cell therapy All these current topics shed light on stem cell technology which may lead to the development of effective clinical modalities for human liver diseases I believe this book will become the gold standard on this topic and will be widely distributed and read by people in many scientific fields, such as cellular biology, molecular biology, tissue engineering, liver biology, cancer biology, and stem cell therapy Tokyo, Japan Takahiro Ochiya v Contents Preface Contributors PART I SEVERAL HEPATIC PROGENITOR CELLS Purification and Culture of Fetal Mouse Hepatoblasts that Are Precursors of Mature Hepatocytes and Biliary Epithelial Cells Nobuyoshi Shiojiri and Miho Nitou Clinical Uses of Liver Stem Cells Yock Young Dan Identification and Isolation of Adult Liver Stem/Progenitor Cells Minoru Tanaka and Atsushi Miyajima Isolation and Purification Method of Mouse Fetal Hepatoblasts Luc Gailhouste Isolation of Hepatic Progenitor Cells from the Galactosamine-Treated Rat Liver Norihisa Ichinohe, Junko Kon, and Toshihiro Mitaka PART II v ix 11 25 33 49 HEPATIC DIFFERENTIATION FROM STEM CELLS Purification of Adipose Tissue Mesenchymal Stem Cells and Differentiation Toward Hepatic-Like Cells Agnieszka Banas Development of Immortalized Hepatocyte-Like Cells from hMSCs Adisak Wongkajornsilp, Khanit Sa-ngiamsuntorn, and Suradej Hongeng Isolation of Adult Human Pluripotent Stem Cells from Mesenchymal Cell Populations and Their Application to Liver Damages Shohei Wakao, Masaaki Kitada, Yasumasa Kuroda, and Mari Dezawa Generation and Hepatic Differentiation of Human iPS Cells Tetsuya Ishikawa, Keitaro Hagiwara, and Takahiro Ochiya 10 Efficient Hepatic Differentiation from Human iPS Cells by Gene Transfer Kenji Kawabata, Mitsuru Inamura, and Hiroyuki Mizuguchi vii 61 73 89 103 115 viii Contents 11 “Tet-On” System Toward Hepatic Differentiation of Human Mesenchymal Stem Cells by Hepatocyte Nuclear Factor Goshi Shiota and Yoko Yoshida 12 SAMe and HuR in Liver Physiology Laura Gomez-Santos, Mercedes Vazquez-Chantada, Jose Maria Mato, and Maria Luz Martinez-Chantar PART III 153 LIVER STEM CELLS AND HEPATOCARCINOGENESIS 14 Identification of Cancer Stem Cell-Related MicroRNAs in Hepatocellular Carcinoma Junfang Ji and Xin Wei Wang PART V 133 BD FORMATION FROM STEM CELLS 13 Transdifferentiation of Mature Hepatocytes into Bile Duct/ductule Cells Within a Collagen Gel Matrix Yuji Nishikawa PART IV 125 163 APPLICATION OF LIVER STEM CELLS FOR CELL THERAPY 15 Intravenous Human Mesenchymal Stem Cells Transplantation in NOD/SCID Mice Preserve Liver Integrity of Irradiation Damage Moubarak Mouiseddine, Sabine Franỗois, Maõmar Souidi, and Alain Chapel 16 Engineering of Implantable Liver Tissues Yasuyuki Sakai, M Nishikawa, F Evenou, M Hamon, H Huang, K.P Montagne, N Kojima, T Fujii, and T Niino 17 Mesenchymal Stem Cell Therapy on Murine Model of Nonalcoholic Steatohepatitis Yoshio Sakai and Shuichi Kaneko Index 179 189 217 225 Contributors AGNIESZKA BANAS • Laboratory of Molecular Biology, Institute of Obstetrics and Medical Rescue, University of Rzeszów, Faculty of Medicine, Rzeszow, Poland ALAIN CHAPEL • IRSN, DRPH/SRBE/LTCRA, CEDEX 92262, France YOCK YOUNG DAN • Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore MARI DEZAWA • Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Japan F EVENOU • Laboratoire Matière et Systèmes Complexes (MSC), Bâtiment Condorcet, Universitộ Paris Diderot, Paris 7, France SABINE FRANầOIS ã IRSN, DRPH/SRBE/LTCRA, CEDEX 92262, France T FUJII • Institute of Industrial Science, University of Tokyo, Tokyo, Japan LUC GAILHOUSTE • Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan LAURA GOMEZ-SANTOS • Metabolomics Unit, CIC bioGUNE, Technology Park of Bizkaia, Bizkaia, Basque Country, Spain KEITARO HAGIWARA • Division of Molecular and Cellular Medicine, National cancer Center Research Institute, Tokyo, Japan M HAMON • Department of Mechanical Engineering, Auburn University, Auburn, AL, USA SURADEJ HONGENG • Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand H HUANG • Okami Chemical Industry Co Ltd, Kyoto, Japan NORIHISA ICHINOHE • Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan MITSURU INAMURA • Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Science, Osaka University, Osaka, Japan TETSUYA ISHIKAWA • Core Facilities for Research and Innovative Medicine, National cancer Center Research Institute, Tokyo, Japan JUNFANG JI • Laboratory of Human Carcinogenesis, Bethesda, MD, USA SHUICHI KANEKO • Center for Liver Diseases, Kanazawa University Hospital, Kanazawa, Japan; Department of Gastroenterology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan KENJI KAWABATA • Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Osaka, Japan MASAAKI KITADA • Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Japan JUNKO KON • Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan N KOJIMA • Institute of Industrial Science, University of Tokyo, Tokyo, Japan ix 16 211 b 8.0 10.0 Albumin production (μg/day/106-cells) Glucose consumption (mg/day/106-cells) a Engineering of Implantable Liver Tissues 7.5 5.0 2.5 0.0 6.0 4.0 2.0 0.0 Culture time (day) 9 Culture time (day) Fig 15 Kinetics of glucose consumption (a) and albumin production (b) during days of perfusion culture of fetal porcine hepatocytes in scaffolds with flow channels Consumption and production are shown per 106 cells immobilized on day the necessity to improve the resolution of fabrication processes A recent advance using a special microsyringe disposition system enabled enhanced resolutions down to several tens of micrometers (39) Such improvements are likely to enable the direct and organized fabrication of the macroporous structure itself, as opposed to the current random process using porogens, in the near future Conclusions In this chapter, we introduced ours and related approaches to liver tissue engineering in three configurations such as thick cellular sheets, macroporous scaffold sheets, and flow channel-containing macroporous scaffolds (Fig 1) Under the current boundary condition that in vitro prevascularization of the engineered tissue is almost impossible, the former two configurations are promising in actual human clinical trials for liver diseases that can be treated by implantation of relatively small tissue constructs The third approach should enable the implantation of liver tissues of a much larger mass in the future In any case, we would like to stress the fact that oxygen supply is the key factor to design and organize liver tissue This enables the cells to utilize aerobic respiration that produces almost 20 times more ATP than anaerobic respiration for the same glucose consumption This also allows the cells to use their maximum reorganization capability that cannot be 212 Y Sakai et al observed in conventional anaerobic conditions One thing we need to consider is the actual oxygen concentration at the cell surface to avoid excess oxidative stress This seems to be very important when we try to obtain mature hepatocytes from stem or progenitor cells The following are remaining important issues to be overcome in the future Expected Future Works 9.1 Formation of a Bile Canaliculi Network In current engineered liver tissues of small mass, bile acids produced by the cells and accumulated in the bile canaliculi leak back to the blood flow before being finally eliminated by the remaining host liver through the host bile duct (3) However, if we really think about substitution of the host liver with engineered liver tissues, we need to arrange a bile canaliculi/duct network over the engineered tissues One clue to the solution is the experimental results by Sudo et al (40, 41) about the functional canaliculi network and transport of bile acids to small bile pools formed in hepatocyte progenitor colonies As a next trial, combination with appropriate microfabrication technologies may give a new insight into organization of such advanced hepatic tissues in vitro 9.2 Endothelialization and Angiogenesis For the third approach where a macroscale flow channel network is to be arranged, because it is finally perfused with the host blood flow upon implantation, complete pre-endothelialization of all the inner surfaces are necessary In addition, further angiogenesis toward the macroporous structure is expected to allow good mass transfer between the cells and the blood flow At present, no one has succeeded in vitro in either complete endothelialization of engineered tissues or formation of a perfusable microvasculature Overall, recent reports in this area demonstrate the necessity of various supporting cells such as fibroblasts or pericytes (or their progenitors) as well as the parenchymal cells of the relevant organ, since those supporting cells promote not only vascular formation (42) but also liver progenitor maturation (43) In vivo, the first system that forms in the embryo is the vascular system, and other organs develop subsequently; it has been shown that liver and pancreas formation in the embryo is promoted by endothelial cells (44, 45) In future, the most efficient way to produce an artificial liver may be to reproduce in vitro the conditions that enable liver formation in the embryo, using embryonic stem cells or induced pluripotent stem cells in prevascularized scaffolds In any case, developmental biology may provide important guiding principles to tissue engineers 16 9.3 Macroscale Oxygenation Engineering of Implantable Liver Tissues 213 When we really target organization of large liver tissues, we need to pay attention to macroscale clean from oxygen depletion in addition to microscale considerations This is the simple mass balance between tissue oxygen consumption and the oxygen amount supplied by the vessel Very low oxygen solubility in culture medium is the first limiting factor; × 10−7 mol O2/mL culture medium under 21% O2 in the gas phase, which is about 1/70th that of blood The second limiting factor is the maximum tolerable shear stress at the inner walls of the flow channels, ~15 dyn/ cm2, which restricts the maximum culture medium flow rate When we think about a typical hepatocyte arrangement around a microvessel, the maximum number of viable hepatocytes along the vessel is calculated to be 24.5 cells (Fig 16), leading to the prediction that the maximum feasible tissue volume is only 50 cm3 Therefore, effective oxygen carriers should be incorporated into the culture medium when we intend to produce large liver tissue equivalents There are two types of oxygen carriers: perfluorocarbon (PFC)based and hemoglobin (Hb)-based carriers PFC is used as a suspension of its emulsions and the maximum concentration of PFC is around 15% (v/v) Therefore, the overall oxygen solubility of such PFC-containing culture medium is at most four times higher than that for culture medium Among the various Hb-based RBC substitutes, polyethyleneglycol (PEG)-decorated liposome encapsulated Hb (LEH) is one of the most promising designs for a RBC Cellular oxygen consumption Maximum oxygen supply rate Diameter of the micro-blood vessel (sinusoid), 10 mm 24.5 cells Oxygen solubility under 21% oxygen partial pressure ϫ Maximal allowable flow rate, 1.47ϫ 10-7 mL/s C = mol/mL at the out let 16.4 mm Maximal allowable shear stress at the inner wall of the micro-blood vessel, 15 dyn/cm2 Hagen-Poiseuille equation Fig 16 Macroscale oxygen mass balance between per minute-based maximum supplied amount and cellular consumption along one blood vessel 214 Y Sakai et al substitute for infusion to humans (46) We therefore checked its toxicity and efficacy in adult and fetal rat hepatocytes Although there was no toxicity in adult rat hepatocytes (47) and the LEH remarkably improved hepatocyte viability and functions in perfusion culture, LEH showed strong toxicity toward fetal hepatocytes LEH incorporated into cells was broken up and released free Hb molecules in the cells, probably causing toxicity via production of reactive oxygen species (ROS), against which fetal cells have not yet developed sufficient defense mechanisms (48) Therefore, Hb-based carriers with improved design will be necessary in the future Acknowledgments The studies were carried out based on various scientific grants, such as Grant-in-Aids for 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Mesenchymal stem cells are pluripotent somatic cells that can differentiate into several cell types, including hepatocytes Moreover, they are obtainable from easily accessible autologous adipose tissue, making them ideal for regenerative therapy This chapter describes experimental methods for isolating mesenchymal stem cells from murine adipose tissues and expanding them, and also describes murine chronic liver disease, steatohepatitis, for the study of experimental regenerative treatments of chronic liver disease Key words: Mesenchymal stem cells, Adipose tissue, Nonalcoholic steatohepatitis Introduction Liver disease is a major health issue worldwide, and includes chronic hepatitis and acute liver failure due mostly to infection by hepatitis or other viruses and drug hepatotoxicity (1) The most intense form of acute liver injury is fulminant hepatitis, which results in rapid and massive destruction of hepatocytes, leading to acute hepatic failure By contrast, the pathological features of chronic liver diseases are characterized by persistent hepatic inflammation and subsequent fibrotic change that distorts the fine lobular architecture of the liver tissue This ultimately leads to end-stage chronic liver injury, which manifests clinically as encephalopathy, due to the failure of various metabolic processes and impaired portal circulation The liver is unique in that hepatocytes per se (2), or progenitor cells (3, 4) can proliferate and restore the original architecture Takahiro Ochiya (ed.), Liver Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol 826, DOI 10.1007/978-1-61779-468-1_17, © Springer Science+Business Media, LLC 2012 217 218 Y Sakai and S Kaneko and function of the liver However, with massive destruction of parenchymal hepatocytes or chronic distortion of the liver architecture with advanced fibrosis the liver cannot regenerate sufficiently The most effective and radical treatment for hepatic failure is liver transplantation However, this is limited by the availability of donors, as there are too few donors compared to the population of hepatic failure patients Even when a donor is available, the relatively high mortality of the transplantation procedure and the permanent requirement for immunosuppressants are major burdens to the recipient Regenerative therapy is a novel alternative treatment to liver transplantation for the severely impaired, malfunctioning cirrhotic liver Bone-marrow stem cells are thought to contribute to liver regeneration (5–8), although it is controversial whether bone-marrow hematopoietic stem cells can differentiate into hepatocytes (9–12) Mesenchymal stem cells are pluripotent somatic stem cells that can differentiate into mesodermal lineage cells, such as adipocytes, chondrocytes, and osteocytes (13), as well as into nonmesodermal lineage cells, such as cardiomyocytes (14, 15) and hepatocytes (16–19) They reside in the bone marrow, umbilical cord, and adipose tissues; adipose tissues are especially rich in mesenchymal stem cells For regenerative cell therapy, autologous cells would be ideal, avoiding the requirement for matching the major histocompatibility antigens to prevent immunological rejection Consequently, bone marrow and adipose tissues are attractive sources of mesenchymal stem cells for regenerative therapy Mesenchymal stem cells may also have favorable biological effects on fibrosis (20, 21) and inflammation (22) This chapter describes experimental methods for studying regenerative therapy for chronic liver disease using mesenchymal stem cells, the culture of mesenchymal stem cells from murine adipose tissue, and a murine model of steatohepatitis that resembles human nonalcoholic steatohepatitis (23) Other methods and their application to liver disease models are also discussed Materials 2.1 Reagents Collagenase type I (Wako Pure Chemical Industries, Osaka, Japan) Phosphate-buffered saline without calcium and magnesium [PBS(−)] (Wako Pure Chemical Industries) DMEM/nutrient mixture Ham F-12 (DMEM/F12) with l-glutamine, 15 mM HEPES (Invitrogen, Life Technologies, Carlsbad, CA) Fetal bovine serum (FBS, Invitrogen) 17 Mesenchymal Stem Cell Therapy on Murine Model… 219 Antibiotic/antimycotic (100×), liquid (Invitrogen) 0.05% w/v trypsin – 0.53 mmol/L EDTA-4Na (Wako Pure Chemical Industries) Pentobarbital sodium (64.8 mg/ml) (Schering-Plough Animal Health, Tokyo, Japan) Atherogenic and high-fat diet (ATH + HF): 38.25% CRF-1 (standard chow, Charles River Laboratories Japan, Yokohama, Japan), 60.0% cocoa butter, 1.25% cholesterol, 0.50% cholate (Oriental Yeast, Tokyo, Japan) Ethanol 10 α-Cyanoacrylate adhesive 2.2 Reagent Preparation Collagenase solution: g of collagenase type I powder is dissolved in 133 ml of PBS and stored at −80°C until use Culture medium: DMEM/F12 supplemented with antibiotic/ antimycotic liquid and 10% heat-inactivated FBS and stored at 4°C Dilute pentobarbital with PBS(−) at tenfold for anesthesia of mice 2.3 Animal 2.4 Equipment C57Bl/6 J mice (male, 8–10 weeks old, Charles River Laboratories, Yokohama, Japan) Operating scissors Tweezers Needle Needle holder 15-ml polypropylene conical tube (BD Falcon, Franklin Lakes, NJ) 100-μm cell strainer (BD Falcon) 6-cm culture dish (Nunc, Rockside, Denmark) 5-0 silk thread (Niccho Industry, Tokyo, Japan) 27-gauge needle with 1-ml syringe Methods 3.1 Isolation and Culture of Murine Mesenchymal Stem Cells from Adipose Tissue All animal experiments should comply with national laws and institutional regulations Euthanize a C57Bl/6J mouse by cervical dislocation Disinfect the skin with 70% ethanol 220 Y Sakai and S Kaneko Make a midline abdominal skin incision and peel off the skin to expose the subcutaneous inguinal region Obtain adipose tissue from the subcutaneous inguinal region by cutting the connective tissues between the adipose tissue and skin, and place in a 6-cm culture dish with PBS(−) Remove the lymph nodes from the adipose tissue using tweezers (see Note 1) Cut the obtained adipose tissue into 1–2-mm pieces with scissors Put the fragmented adipose tissue in a 15-ml conical tube containing 10 ml of PBS Centrifuge it at 200 × g for and remove the supernatant 10 Add 10 ml of PBS (−) to the tube and centrifuge it at 200 × g for 11 Remove the supernatant as in step 12 Put the PBS(−)-rinsed adipose tissue fragments into a 15-ml conical tube with 2–3 ml of collagenase aliquot 13 Incubate the adipose tissue fragments and collagenase with shaking at 37°C in thermostat bath for h 14 Add an equal volume of DMEM/F12 containing 10% heatinactivated FBS supplemented with 1% antibiotic/antimycotic liquid 15 Centrifuge at 200 × g for 10 16 Remove the debris and PBS(−) 17 Resuspend the remaining cells in PBS(−) and filter them through a 100-μm cell strainer 18 Centrifuge at 200 × g for 10 19 Remove the PBS(−), suspend the cells in ml of DMEM-F12 supplemented with heat-inactivated FBS, and place in a 6-cm culture dish (Fig 1a) 20 Replenish the culture medium with fresh complete medium the next day 21 Replenish the medium every 3–4 days The culture usually reaches 70% cell confluence after 10 days (Fig 1a) 22 Cells can usually be passaged and expanded eight or nine times until morphological change appears (Fig 1b) (see Notes and 3) 3.2 Establishing a Murine Steatohepatitis Model C57Bl/6J male mice are maintained in colony cages with a 12-h light/12-h dark cycle 8-week-old mice are fed an ATH + HF diet for 24 weeks The livers of these mice develop steatosis in hepatocytes accompanied with pericellular fibrosis (Fig 2a, b), resembling the liver histology seen in advanced nonalcoholic steatohepatitis (23) 17 Mesenchymal Stem Cell Therapy on Murine Model… 221 Fig The appearance of cultured cells obtained and expanded from murine adipose tissues (a) The characteristic “spindle shape” of the mesenchymal stem cells is observed (b) Morphological change of cells appeared usually after ten passages (c) CD105 expression of cultured cells (eight passages) Fig Histology of the liver obtained from mice, which were fed with ATH + HF diet for 24 weeks (a) HE staining (×100), (b) AZAN staining (×100) 3.3 Experimental Therapeutic Application of Mesenchymal Stem Cells in the Murine Steatohepatitis Model Mice that develop steatohepatitis on the ATH + HF diet for 24 weeks are anesthetized by an intraperitoneal injection of 200 μl of diluted pentobarbital Expanded mesenchymal stem cells isolated from murine adipose tissues are prepared A midabdominal incision is made and the middle lobe of the liver is exposed A 2–3-mm liver specimen is obtained by cutting with scissors and the cut area is closed using α-cyanoacrylate adhesive After the biopsy, a × 105/200 μl mesenchymal stem cell aliquot is injected into the subcapsule of the spleen using a 27-gauge needle with a 1-ml syringe The peritoneum and skin are sutured with 5-0 silk The mice are kept on the ATH + HF diet for two more weeks 222 Y Sakai and S Kaneko After weeks, the mice are euthanized by cervical dislocation Serum and liver tissues are collected, and RNA is extracted from the liver tissues These samples are assayed to assess the therapeutic effect of administering mesenchymal stem cells (see Notes and 5) Notes This step is required to avoid contamination of mesenchymal stem cell culture by resident lymphocytes The method of isolating and culturing mesenchymal stem cells from adipose tissues is described Mesenchymal stem cells also reside in bone marrow, and the methods for isolating and expanding mesenchymal stem cells from bone marrow tissues have been reported (24, 25) The latter report states that mouse mesenchymal stem cells were isolated by aspiration of bone marrow in the tibia and femur, and cultured in DMEM supplemented with 15% FBS The culture medium was replenished frequently With this method, confluent mesenchymal stem cells can be obtained after 21 days CD105 is a marker for mesenchymal stem cells Using the CD105 MultiSort Kit (Miltenyi, Auburn, CA), the mesenchymal cell fraction can be enriched To assess the effect of mesenchymal stem cells on the liver in the steatohepatitis murine model, real-time quantitative PCR expression analysis of liver RNA samples was performed Compared to the pretreatment level, expression of interleukin-6 was upregulated and that of interleukin 15 receptor alpha was downregulated after the mesenchymal stem cell treatment (unpublished observation) The carbon tetrachloride (CCl4)-induced chronic liver disease model is another model of chronic liver disease (5, 26) that can be used for experimental regenerative therapy To establish this murine model, C57Bl/6 mice are intraperitoneally injected with ml/kg of CCl4 for weeks These mice develop advanced fibrotic changes in the liver, i.e., cirrhosis The therapeutic effect of mesenchymal stem cells on chronic liver disease can also be studied using this model It is reported that entire fractions of bone marrow cells can improve liver fibrosis in this CCl4-induced cirrhotic murine model, presumably via the activation of matrix metalloproteinase (5) The rat is an alternative rodent for establishing chronic liver injury models, either CCl4induced (27, 28) or steatohepatitis (29) cirrhosis 17 Mesenchymal Stem Cell Therapy on Murine Model… 223 References Williams R (2006) Global challenges in liver disease Hepatology;44:521–526 Michalopoulos GK, DeFrances MC (1997) Liver regeneration Science;276:60–66 Evarts RP, Nagy P, Nakatsukasa H et al (1989) In vivo differentiation of rat liver oval cells into hepatocytes Cancer Res;49:1541–1547 Dorrell C, Grompe M (2005) Liver repair by intra- and extrahepatic progenitors Stem Cell Rev;1:61–64 Sakaida I, Terai S, Yamamoto N et al (2004) Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice Hepatology;40:1304–1311 Terai S, Ishikawa T, Omori K et al (2006) Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion 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stem cells from mouse bone marrow Nat Protoc;4:102–106 Natsume M, Tsuji H, Harada A et al (1999) Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice J Leukoc Biol;66: 601–608 Oyagi S, Hirose M, Kojima M et al (2006) Therapeutic effect of transplanting HGFtreated bone marrow mesenchymal cells into CCl4-injured rats J Hepatol;44:742–748 Hardjo M, Miyazaki M, Sakaguchi M et al (2009) Suppression of carbon tetrachlorideinduced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow Cell Transplant;18:89–99 Ota T, Takamura T, Kurita S et al (2007) Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis Gastroenterology;132:282–293 INDEX A F Adenovirus vector .104, 109, 116, 118–119 Adipose tissue-derived mesenchymal stem cells (AT-MSC) 61–65 Alanine amino-transferase (ALT) 182–186 ALT See Alanine amino-transferase (ALT) Antibody 5, 27, 28, 30, 31, 40, 46, 55, 56, 76, 83, 84, 91, 92, 94, 95, 100, 101, 107, 112, 143–145, 165, 167, 195 Aspartate amino-transferase (AST) 182–186 AST See Aspartate amino-transferase (AST) Flow channel 190 -cytometry 26, 30, 31, 83, 85, 96, 101 Fluorescence-activated cell sorting 91, 164 G Galactosamine .49–57 H CD44 50, 52–57 Cell immortalization cell interaction .4 sorting 31, 34, 35, 37, 38, 40, 41, 43–46, 167, 172 Cellular sheet 211 Chronic liver diseases 89, 90, 154, 217, 218, 222 c-Myc .103, 105, 106, 109, 110, 112 CYP450 73, 74, 76, 81, 82, 84 Hepatic cancer stem cells differentiation 61–71 progenitor cells 49–57 Hepatoblasts 3–9, 33–46, 115, 116, 119–120, 154 Hepatocellular carcinoma 127, 135, 163–174 Hepatocyte differentiation 12, 16, 19, 89, 90, 108, 115, 116, 138–141, 145 like cell 15, 73–82 proliferation 135–137, 153, 187 transplantation 11, 13–15, 17, 18 HNF3b 129 Human iPS cells .103–123 MSC 73–86, 125–131, 179–187 HuR .133–146 D I Differentiation 3, 16, 34, 61–71, 75, 90, 103–123, 138, 154, 163, 183, 206 Drug development metabolism 73 Inflammatory cytokines 153 iPS cells See Pluripotent stem cells (iPS) Irradiation exposure 186 B Bile duct cells 153–160, 184, 186, 212 Bioartificial liver assisted device 19–20 Bipotent stem cells 33 C E K KLF4 103, 105, 106, 109, 110, 112 E-cadherin 4, 5, 7, 8, 34, 35, 37, 38, 40–46 Embryonic stem (ES) cells 15, 62, 90, 103, 140, 206, 212 Epithelial cell adhesion molecule (EpCAM) 26–28, 30, 31, 163–165, 171, 172 Extracellular matrices 3, 154 L Liver bilic acids 182 damages 25, 26, 89–101, 186 Takahiro Ochiya (ed.), Liver Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol 826, DOI 10.1007/978-1-61779-468-1, © Springer Science+Business Media, LLC 2012 225 LIVER S 226 Index TEM CELLS : METHODS AND PROTOCOLS disease 11, 12, 18, 19, 33, 89, 90, 153, 154, 211, 217, 218, 222 fibrosis 153, 218, 222 progenitor cells 3, 140 regeneration 25, 135, 136, 140 stem cells, M Macroporous scaffold 193, 205–207, 209–211 MACS See Magnetic cell sorter (MACS) Magnetic cell sorter (MACS) 5, 51, 52, 55, 62, 63, 65, 69, 70 Mesenchymal stem cell (MSC) 14, 61–71, 73–86, 125–131, 140, 179–187, 217–222 Methionine adenosyltransferase (MAT) 134–138, 142 MicroRNA microarray 165–166, 168–169 MicroRNAs 163–174 Mouse fetal hepatoblasts (MFH) 33–46 Muse cells 90–96, 98–100 N NOD/SCID mice 164, 171, 179–187 Non-alcoholic steatohepatitis 135, 217–222 Nonparenchymal cells 3–7, 9, 19, 26, 45, 49, 156, 195, 196 O Oct3/4 .90, 98, 103, 105–110, 112 Oval cells 25, 26, 28, 31, 33, 42, 49, 53, 55–57, 89, 90 Oxygen .190–192, 197–209, 211–214 P Plasticity 154 Pluripotent stem cells (iPS) 15, 18–21, 89–101, 103–123 Polymerase chain reaction (PCR) 99, 106, 108, 110–113, 129, 145, 166, 170, 173, 182 Q Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 143, 164 R Regenerative medicine 180 Respiration 202, 204, 211 Retrovirus 103, 104, 109, 127 S S-Adenosylmethionine (SAMe) 133–146 Small hepatocytes 49 Sox2 90, 98, 103, 105, 106, 109, 110, 112 Spheroid assay 164, 165, 171 SSEA–3 See Stage-specific embryonic antigen–3 (SSEA–3) Stage-specific embryonic antigen–3 (SSEA–3) 90–92, 94–96, 98, 100, 101, 105, 111 Stem cells Stem/progenitor cells .15, 25–31, 49, 56, 89, 141, 146 T Tetracycline operator 127 inducible system 125 Tet repressor 125, 127, 130, 131 Therapeutic transplantation 179 Three-dimensional cultures (3D culture) 18, 154, 159, 190, 196, 205, 206 Thy1 53–57, 105, 111 Toxicology 12, 73 Transdifferentiation 14, 15, 153–160 Transfection .78–80, 91, 94, 104, 109, 128–129, 143, 144, 164, 166, 170–171, 173 Tumorigenicity assay 164, 165, 171 ... of stem cells in repair of liver injuries Several types of stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, haematopoietic stem cells, and mesenchymal stem cells, ... Preface A Brief Outline of the Aims and Target Audience of Liver Stem Cells The role of a putative stem cells and liver- specific stem cell in regeneration and carcinogenesis is reviewed in this... formation from stem cells; (4) Liver stem cells and hepatocarcinogenesis; and (5) Application of liver stem cells for cell therapy All these current topics shed light on stem cell technology